This is a renewal application for the RO1 study ?A longitudinal immunological study for ME/CFS biomarker discovery?. We will recruit and follow-up new cases and a sample of cases of the UK ME/CFS cohort, focusing on detailed immunological and clinical phenotypes and analyses of their inter-relationships. Participants and longitudinal assessments: 110 ME/CFS cases ( severe, mild-moderate) meeting all of the CDC-19942, Canadian1 and IOM4 criteria will be assessed every 6 to 12 months for 5 time-points, enabling the mapping of changes in biomarker expression onto clinical parameters (and vice versa); to precisely stratify different case types; and to accurately analyse biomarkers at different stages of disease progression and their time-relationship with clinical parameters. Activities and objectives: In a prospective cohort study, we will: i) recruit and follow up a total of 110 ME/CFS cases, of which at least 100 cases will have complete data and sample sets; ii) stratify patients according to trends in symptom severity, a) ?improving?, b) ?stable? or c) ?worsening?, using scores related to pain, fatigue and functional status39. For in-depth immunological profiling, we will: i) conduct detailed ex vivo phenotyping of PBMC populations by flow cytometry; ii) measure the functional response of NK and T cells after in vitro stimulation by flow cytometry; iii) analyze secreted cytokines in supernatants of stimulated PBMC cultures by multiplex bead array; and, iv) genotype donors for MHC Class1 and KIR to inform analysis of flow cytometry data. To integrate analyses and map immunological data onto clinical data and determine whether changes in immune parameters precede, follow, or predict the clinical trajectory, we will: i) quantify correlations between immunological biomarkers and ii) identify biomarkers associated with changes in ME/CFS clinical status. We will consider patients? perspectives, ethical, legal, societal issues at all stages. To ensure quality and maximum research output we will continue using the UK ME/CFS cohort procedures and protocols during the longitudinal follow up of patients. We will follow standardised clinical, data and samples handling and laboratory procedures in the follow up of ME/CFS patients meeting specific clinical criteria. Innovation and Strategic Need: This is, to our knowledge, the first long-term prospective study of ME/CFS to incorporate both ambulatory and severe cases and to include comprehensive clinical data and in-depth immunological profiling. The study is unique for its strict inclusion criteria and detailed clinical phenotyping, adding specificity and validity to our analyses. In the long term, identification of robust biomarkers will allow the correlation of ME/CFS phenotype with disease severity and prognosis and may reveal new options for interventions. Because 1- 2.5 million Americans have ME/CFS4, this study has the potential to improve the lives of a large patient population in the U.S. as well as advance the state of the field in the U.S. and internationally. Patients and stakeholders are involved in all aspects of the research.